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Mutational analysis of the Rieske iron-sulfur protein (ISP) has provided useful information on how the ubiquinol:cytochrome c oxidoreductase complex functions to transfer electrons through the complex as well as transport protons from the matrix space to the intermembrane space. Mutations of amino acid residues in the conserved hinge region of ISP have resulted a general decrease of enzymatic activity by 30–70%. Recent developments in crystallography have helped show that the aqueous head region of ISP is mobile. Characterization of the hinge region mutants combined with molecular dynamics simulations of the X-ray crystal structure show that one reason for the conservation of the hydrophobic hinge region residues is to prevent steric interaction among side chains that would impede the movement of the head region. A compensatory double mutation has been generated that alleviates the steric strain induced by a point mutation, restoring enzymatic activity to wild type levels. Mutations in the solvent-exposed head region have also had deleterious effects on enzymatic activity, even when those mutations were far removed from the [2Fe2S] cluster that is the active metal center for the protein. Closer examination has revealed a complex network of hydrogen bonds within the head region that stabilizes the structure of the head region and the orientation of the [2Fe2S] cluster. Mutations that severely disrupt this network either prevent stable assembly of ISP into the bc1 complex or prevent proper motion of the head group between the b-type and c1-type hemes. More conservative mutations reduce enzymatic activity by about 50% with negligible effect on proton pumping. Molecular dynamics studies predict the effects of the mutations on the enzyme structure. Conservative mutations have been generated that allow wild type levels of enzymatic activity, though some change the local scheme of hydrogen bonding. The bc1 complex has been thought to be the site of generation of significant quantities of reactive oxygen species (ROS). Studies were undertaken with mutants known to impede ISP mobility to determine if such mutants contributed to an increase in ROS production.